Liposomes are well established nanoparticles that can enhance the efficacy of therapeutically active drugs by improving the plasma distribution and pharmacokinetics of the drugs over non-encapsulated forms (e.g., Weinstein, Liposomes: From Biophysics to Therapeutics, (Ostro, M. J., ed.), Marcel Dekker, Inc., N.Y., pp. 277-338, (1987). For example, Vincristine Sulfate Liposome Injection (VSLI) is a liposome formulation of the anti-cancer therapeutic vincristine sulfate encapsulated in sphingomyelin-cholesterol liposomes which provides greater efficacy than standard vincristine sulfate injection USP (VSI). Clinical trials have also shown that VSLI facilitates dose intensification by significantly extending vincristine's circulation half-life compared to non-encapsulated vincristine. The liposome provides the mechanism for delayed drug release and the liposome size allow the drug to accumulate in cancer tissues by extravasation (Webb et al., Cancer Chemother. Pharmacol 42:461-470, 1998; Shan et al., Cancer Chemother. Pharmacol 58:245-255, 2006). These features translate into improved clinical benefit over the standard VSI.
Encapsulation of vincristine sulfate into sphingomyelin-cholesterol liposomes is typically achieved by using an acidic intraliposomal pH (e.g., pH of 4) and an exterior medium at a neutral pH (e.g., pH of 7). This pH gradient allows the weakly basic vincristine to diffuse into the liposome interior with high efficiency (Cullis et al., Trends in Biotech 9: 268-272, 1991; Boman et al., Bioch Biophys Acta, 1152:253-258, 1993). In order for vincristine to accumulate in the liposome interior with the transmembrane pH gradient, the liposome membrane must become temporarily permeable to the steric bulk of vincristine. Thus, unlike neutral or anionic drugs that often can be passively encapsulated into liposomes, the temperature of the sphingomyelin-cholesterol liposome must be increased in order for the transmembrane pH gradient to function with vincristine. The liposome bilayer, which is an orientation of interlocking sphingomyelin and cholesterol molecules, requires a unique transient heat pattern to create thermotropic disorder transition states. These transition states essentially abate the weak intermolecular bonding between the membrane lipids, creating gaps in interlocking lipids and allow the liposome biolayer to become temporarily permeable. The encapsulation process takes advantage of the spontaneous self reassembly of the sphingomyelin-cholesterol lipids that occurs on cooling back to ambient temperature, which restores the membrane integrity.
This heating profile for drug encapsulation must be balanced with the chemical instability of vincristine to heat exposure (Vendrig et al., Internatl. J. of Pharmaceutics 50:189-196, 1989; Sethi et al., Cancer Res. 45:5386-5389, 1985). Vincristine is thermally labile and readily degrades to N-desformylvincristine in the presence of elevated temperatures. This formamide hydrolysis of vincristine is a well known degradation pathway and affects the stability shelf life of vincristine sulfate injection (VLSI). For example, VLSI solutions cannot be heat sterilized due to this heat lability and must be stored and shipped at refrigerated temperature to realize extended stability.
Accordingly, at the present time Vincristine Sulfate Liposome Injection (VSLI) is prepared from the individual components at a pharmacy according to the directions provided on the FDA-approved label (www.accessdata.fda.gov; Reference ID: 3172211, 2012). These directions include a heating procedure that requires the use of a water bath in order to achieve efficient encapsulation of vincristine in the sphingomyelin-cholesterol liposomes and maintain chemical purity of vincristine. The excellent heat transfer properties of water allow greater than 95% encapsulation of vincristine with no appreciable chemical degradation of the drug.
Since VSLI is an injectable drug, the manufacture of the components and pharmacy preparation are strictly regulated to maintain sterility. Accordingly, the use of an open water bath during preparation of VSLI requires additional resources, planning, and equipment (e.g., floating ring), including an aseptic hood or “clean” room in order to maintain an aseptic environment. In some pharmacies, the constitution of VSLI cannot be done due to the restrictions on maintaining a sterile environment.
Accordingly, there remains a need for improved methods of preparing VSLI that can be efficiently and reproducibly carried out without the additional resources and equipment currently required.